We propose a novel strategy to align electron microscope (EM) images of single biological particles that employs multiple heavy atom clusters, rigidly fixed to specific locations on the particles, as alignment markers. The heavy atom clusters, which are individually visible and can be precisely localized in EM images, will allow accurate determination of particle position, homogeneity, and orientation. The method should enable rapid structure determination of most biological particles in their native, uncrystalline state and under physiologic conditions, even in the presence of contaminants such as deformed or substoichiometric particles, or particles in alternative conformations. Our goals for this project are to demonstrate the feasibility of the method and to create the reagents and computational tools needed for general use. In preliminary work, we have developed computer software to simulate the method, we have established a requirement for four clusters bound to the protein of interest, we have estimated the degree of immobilization of the cluster necessary for high resolution analysis, we have prepared a single chain antibody Fv fragment (scFv) directed against a suitable model protein and derivatized the scFv with a large gold cluster, and we have determined conditions for imaging the cluster with the retention of high resolution information. Specific aims for the project period are: (1) Optimization of the scFv-cluster conjugate. This will entail site-directed mutagenesis of the scFv combined with variation of the cluster chemistry to achieve the rigid fixation of the cluster required for high resolution analysis. (2) Application to a model protein of known structure. Comparison of results from the proposed method with those obtained for the same protein by Xray crystallography will provide a test of the method and show the resolution that can be obtained. (3) Construction of a modified scFv library, as well as computational tools, for use of the method by the biomedical research community.